Category Archives: Wines and Oenology

The vine growers believe that the land on which they grow vines gives the wines a unique quality, and that is called terroir. We can consider that the physiological response of the vines to the type of soil and climatic conditions, together with the characteristics of the variety and form of cultivation, result in a wine organoleptic properties that define their terroir (Zarraonaindia et al 2015 ). However, it is not known if there could be a very specific microbiota of each terroir, as this subject has been barely studied.

Wine microorganisms in the grapes? Saccharomyces is not there or it has not been found there

The main protagonists of wine fermentations, alcoholic one (yeast Saccharomyces cerevisiae) and malolactic one (lactic acid bacteria Oenococcus oeni) usually do not appear until the must grape is fermenting to wine, in the cellar. In normal healthy grapes, S. cerevisiae is hardly found.

Oenococcus oeni in the grapes ? We have found it !

Regarding O. oeni, so far very little has been published about its presence and isolation from the grapes. In some works, as Sieiro et al (1990), or more recently Bae et al (2006), some lactic acid bacteria (LAB) have been isolated from the surface of grapes, but not O. oeni. Only Garijo et al (2011) were able to isolate a colony (only one) of O. oeni from Rioja grapes. Moreover, DNA of O. oeni has been detected in a sample of grapes from Bordeaux (Renouf et al 2005, Renouf et al 2007) by PCR-DGGE of rpoB gene, although in these works no Oenococcus has been isolated.

I am pleased to mention that recently our team have managed to isolate O. oeni from grapes, and typify them, and we are now working on a publication about it (Franquès et al 2015). Indeed, our research team of lactic acid bacteria (BL-URV), together with colleagues working on yeasts from the same group “Oenological Biotechnology” (Faculty of Oenology at the Universitat Rovira i Virgili in Tarragona, Catalonia, Spain) is working on a European project, called “Wildwine “(FP7-SME-2012 -315065), which aims to analyse the autochthonous microorganisms of Priorat area (South Catalonia), and select strains with oenological potential. This project also involves the Priorat Appellation Council and the cellar Ferrer-Bobet, as well as research groups and associations wineries from Bordeaux, Piedmont and Greece. In the framework of this project we took samples of grapes (Grenache and Carignan) from several vineyards of Priorat (Figure 1), as well as samples of wines doing malolactic fermentation. From all them we got 1900 isolates of LAB. We optimized isolation from grapes from the pulp and juice with various methods of enrichment, and so we got 110 isolated bacteria from grapes, identified as O. oeni by specific molecular techniques. Once typified, we have found that the molecular profiles of these strains do not coincide with commercial strains and so they are autochthonous. In addition, some of these strains from grapes were also found in the corresponding wine cellars.

The grapes have a complex microbial ecology, including yeasts, mycelial fungi and bacteria. Some are found only in grapes, such as parasitic fungi and environmental bacteria, and others have the ability to survive and grow in wines: especially yeasts, lactic acid bacteria (LAB) and acetic acid bacteria. The proportion of all them depends on the maturation of the grapes and the availability of nutrients.

When the fruits are intact, the predominant microbiota are basidiomycetous yeasts as Cryptococcus and Rhodotorula, but when they are more mature, they begin to have micro fissures that facilitate the availability of nutrients and explain the predominance just before the harvest of slightly fermentative ascomycetes as Candida, Hanseniaspora, Metschnikowia and Pichia. When the skin is already damaged more damaging yeasts may appear, as Zygosaccharomyces and Torulaspora, and acetic acid bacteria. Among the filamentous fungi occasionally there may have some very harmful as Botrytis (bunch rot) or Aspergillus producing ochratoxin. Although they are active only in the vineyard, their products can affect wine quality.

On the other hand, environmentally ubiquitous bacteria have been isolated from the grapes skin, as various Enterobacteriaceae, Bacillus and Staphylococcus, but none of them can grow in wine (Barata et al 2012).

Coming back to the possible specific microbiota of terroir, it has been found that some volatile compounds contributing to the aroma of the wine, such as 2-methyl butanoic acid and 3-methyl butanol, are produced by microorganisms isolated in the vineyards, as Gram-positive bacterium Paenibacillus, or the basidiomycetous fungus Sporobolomyces or the ascomycetous Aureobasidium. Therefore, there could be a relationship between some of the microbial species found in grapes and some detected aromas in wine, coming from the must of course (Verginer et al 2010).

Metagenomics as analytical tool of microbiota from grapes

Since conventional methods of isolation and cultivation of microorganisms are slow, laborious and some microbes cannot be grown up in the usual isolation media, massive sequencing methods or metagenomics are currently used. These consist of analysing all the DNA of a sample, and deducing which are the present microorganisms by comparing the sequences found with those of the databases. For bacteria the amplified DNA of V4 fragment from 16S RNA gene is used (Caporaso et al 2012).

This technique has been used with samples of botrytized wines (Bokulich et al 2012) and various LAB have been found (but not Oenococcus), including some not normally associated with wine. It has also been used to see the resident microbiota in wineries and how it changes with the seasons, resulting that in the surfaces of tanks and machinery of the cellar there is a majority of microorganisms neither related with wine nor harmful (Bokulich et al 2013).

With this technique Bokulich et al (2014) have also analysed the grapes and they have seen clear differences between the proportions of bacterial groups (and fungi) from different places, different varieties, as well as environmental or bio geographical conditions. For example, when analysing 273 samples of grape musts from California, the 3 varieties (Cabernet, Chardonnay and Zinfandel) are quite discriminated in a principal components analysis with respect to the bacterial communities found in each sample (Figure 2).

Thus, the dominant bacterial taxa or groups in a variety or given environment could provide some specifics traits on those wines, and this could explain some regional or terroir patterns in the organoleptic properties of these wines (Bokulich et al 2014).

We have also carried out a massive sequencing study with the same grape samples from which we have obtained isolates of O. oeni, as said before (Franquès et al 2015), and in more than 600,000 analysed sequences of 16S rRNA, we have found mainly Proteobacteria and Firmicutes. Among these gram-positive, we have found sequences of lactic acid bacteria (15%) and from these we have successfully confirmed the presence of O. oeni in 5% of the sequences. Therefore, we have isolated O. oeni from grapes and we have detected their DNA in the samples.

The bacterial microbiota of the vineyards and soil

As we see, microbiota of grapes and wine has been studied a little, but the soil microbiota has not been characterized. This one can define more clearly the terroir, which is influenced by the local climate and characteristics of the vineyard.

In Figure 3 the main genera found in different parts of the vine and soil are summarized (Gilbert et al 2014).

Recently an interesting scientific work (Zarraonaindia et al 2015) has been published on this subject, with the aim to see if the soil could be the main original source of bacteria that colonize the grapes. These authors took samples of soil, roots, leaves, flowers and grapes from Merlot vines, from different areas and years, of Suffolk, New York, and they analysed the bacterial DNA by 16S rRNA sequencing. They found that 40% of the species found were present in all samples of soil and roots, while there was more variability in leaves and fruits, and moreover, 40% of those found in leaves and fruits were also found in soils. All this suggests that many bacteria originate in the soil.

Regarding the type of bacteria, they found that Proteobacteria (especially Pseudomonas and Methylobacterium) predominated (Figure 4), mainly in the aerial parts of the plant. There were also Firmicutes as expected, and Acidobacteria and Bacteroides.

Figure 4. Composition of the bacterial community, at Phylum level, in samples from different organs of the vine and its soil (Zarraonaindia et al 2015).

Although variations were observed in all samples depending on the year (there may be different climatic conditions) and according to different edaphic factors (pH, C: N, humidity), the principal-components analysis (Figure 5) showed that the main types of samples (soil, roots, leaves, grapes) differ quite well, and bacterial taxon composition in samples of grape juice before fermentation is similar to that of grapes.

This suggests that the bacterial community found in grapes remains relatively stable until the processing to musts, and that it is more stable than the differences between organs. At the same time, a large number of representatives of bacterial phyla of the grapes come from the soil. This can be explained because when grapes are harvested by hand, they are often placed in boxes that are left on the ground, or for mechanical harvest, the machinery used removes the soil and generates dust, which can colonize the grapes.

Therefore, the soil microbiota is a source of bacteria associated with vines and may play a role in the must and therefore in the wine, and potentially in the formation of the terroir characteristics. Some of these bacteria may have some roles not yet known in productivity or disease resistance of the plant, or contribute to the organoleptic characteristics of wine (Zarraonaindia et al 2015).

In addition, and thinking in wine microorganisms responsible for fermentations, as said, in our laboratory we have confirmed that there are some O. oeni strains in grapes and we have confirmed this by detecting their DNA in the same grapes.

It is really surprising, but it seems so: Italian and Austrian researchers have published a paper (Campisano et al. 2014) which shows that the bacterial species Propionibacteriumacnes, related to human acne, can be found as obligate endophytes in bark tissues of Vitis vinifera, the grapevine.

Some bacterial pathogens of humans, such as Salmonella, are able to colonize plant tissues but temporarily and opportunistically (Tyler & Triplett 2008). In fact, there is a temporary mutual benefit between plants and bacteria, so some of these enterobacteria pathogenic to plants do not live endophytically and can be beneficial for them. These pathogens to humans, in its life cycle, use plants as alternative hosts to survive the environment, passing to the plants through contaminated irrigation water. Therefore, some bacteria are often temporary endophyte guests of plants.

But on the other hand, there are relatively rare cases of bacteria changing the host and adapting to the new host, finally being endophytes. This horizontal transfer happens mostly between evolutionarily close hosts, such as symbiotic bacteria of aphids (insects), which has proven to transfer to other species of aphids (Russell & Moran 2005). It has also been suggested the horizontal transfer of beneficial lactic acid bacteria (Lactobacillus reuteri) in the intestinal tract of vertebrates, since strains of this L. reuteri are similar in several species of mammals and birds.

Well, going beyond, the work of Campisano et al. subject of this review, concludes that bacteria associated with human acne should have passed on the vine, that is, the bacteria would have made a horizontal transfer interregnum, from plants to mammals.

Propionibacterium acnes type Zappae

Acne, as you know, is a common human skin disease, consisting of an excess secretion of the pilosebaceous glands caused by hormonal changes, especially teenagers. The glands become inflamed, the pores obstructed and scarring appears. The microorganism associated with these infections is the opportunistic commensal bacterium P. acnes, a gram-positive anaerobic aero tolerant rod, which fed fatty acids produced by the glands.

Interestingly, other species of the same genus Propionibacterium well known in microbial biotechnology industry are used for the production of propionic acid, vitamin B12, and the Swiss cheeses Gruyere or Emmental.

Campisano et al. have made a study of the vineyard endomicrobioma by the sequencing technique (Roche 454) amplifying the V5-V9 hyper variable region of the bacterial 16S rDNA present in the tissues of vine. In 54 of the 60 plants analyzed, between 0.5% and 5% of the found sequences correspond to the species Propionibacterium acnes. This observation has been confirmed by fluorescent in situ hybridization (FISH) with fluorochromes and specific probes of P. acnes.

Location of P. acnes (fluorescent blue spots) in the bark of a vine stem, seen with FISH microscopy with specific probes for this bacterium (Campisano et al 2004).

The authors of this work proposed for this bacterium the name of P. acnes Zappae, in memory of the eccentric musician and composer Frank Zappa, to emphasize the unexpected and unconventional habitat of this type of P. acnes.

To solve this riddle, Campisano et al. have taken the 16S rDNA sequences and from other genes (recA and tly) from these strains of P. acnes Zappae found in vine and have compared with those P. acnes of human origin in databases. Comparing phylogenies and clusters deducted from them, these researchers have concluded that P. a. Zappae has diversified evolutionarily recently. Studying in detail the recA gene sequences of P. a. Zappae, and taking into account the likely mutation rate and generation time (about 5 hours), they deduce that the diversification from other P. acnes occurred 6000-7000 years ago.

This date coincides with the known domestication of the vine by humans, which is believed to have occurred about 7000 years ago in the southern Caucasus, between the Black Sea and the Caspian Sea, the area of modern Turkey, Georgia, Armenia and Iran (Berkowitz 1996). The vineyard has its origins in a wild subspecies of Vitis that survived the Ice Age and was domesticated. This plant came out to three subspecies, and one of them, Vitis vinifera pontica, spread in the mentioned area and further south in Mesopotamia and then to all south Europe thanks to the Phoenicians.

Therefore, the conclusion is that P. acnes Zappae originated from human P. acnes 7000 years ago, by contact of human hands with grapes and other parts of the vineyard during the harvest and carrying them. As the authors say, this case would be the first evidence of horizontal transfer interregnum, from humans to plants, of a obligate symbiotic bacterium. This also makes more remarkable the adaptability of bacteria. Their ability to exploit new habitats can have unforeseen impacts on the evolution of host-symbiont relationship or even host-pathogen.

First of all, I apologize to friends of this blog for these last five months without any post. Sorry, I had too much work of teaching and research, and also I had to prepare this Conference that I am commenting now.

CEICS is the strategic aggregation, driven by the Universitat Rovira i Virgili, of various institutions and structures of teaching, research, transference, and the productive sector of southern Catalonia, with the goal of becoming an international benchmark in 5 areas: Chemistry, Nutrition-Health, Tourism, Culture-Heritage and Oenology.

In 2012 I had the honor of being named coordinator of subcampus Oenology of CEICS, by the rector of the URV Xavier Grau, who is the president of CEICS.

The subcampus Oenology includes URV, Fundació URV, the technological park VITEC of Falset, IRTA, INCAVI, the cluster INNOVI, and most of catalan DO (Appelation of Origin), especially those of Tarragona, and companies and wineries, such as Freixenet and others.

One of the main objectives of CEICS is to promote the visibility of research done in the region. Therefore, since the beginning of CEICS, with the occasion of the 1st Forum held in November 2011, a conference-meeting like this was already planned, in order to share and diffuse the research done in Catalonia.

Inauguration of the Conference, with the rector of the URV Xavier Grau and the Dean of the Faculty of Oenology Joan Miquel Canals.

In Catalonia we have often the opportunity to attend various scientific conferences or technical meetings on oenology and/or viticulture. The INCAVI, the Catalan Association of Winemakers (ACE) or the cluster INNOVI, or the Facultat d’Enologia from URV often organize special courses or seminars. All of them are very interesting and necessary, and add value to the Catalan wine industry, but perhaps there had not yet been done so far a meeting of all catalan researchers in this field, in order to see all the research that is being carried out, not just in a specific focus or subject.

All in all, we considered appropriate to take the energy and push of CEICS for organizing this conference. The main objective was to publicize and disseminate the research carried out in Catalonia in Oenology and Viticulture and related issues. Before the conference, we reviewed databases of scientific publications in the last years, and we have seen that in Catalonia there are about 20 research groups whose main lines are related to the sciences of oenology and viticulture, but in addition there are 40 other groups and researchers that although his main area of research is not the viticulture and oenology, in recent years they have published several papers related to aspects of vitiviniculture, even from scientific disciplines apparently far away. All they were invited to this conference held in Tarragona (at Campus Sescelades URV), to present their most innovative research in the form of posters. I must thank them all for the good response, since there was a total of 44 posters presented.

Another implicit goal of the conference was meeting people. This conference was an opportunity to meet, discuss and explore the details of recent work with colleagues from different centers in Catalonia, maybe even meeting in person for the first time in some cases, and promote collaboration between groups.

But in addition, this Conference has not been a meeting exclusively for scientists. The research is meaningless if it has no practical application and benefits in the winemaking process, though often not immediate implementation. It binds with the main objective of the conference, as mentioned, to make visible the research done here. In this regard, some of the posters that were presented have been selected so that the authors briefly commented on the general sessions, and the selection has been done keeping in mind the interest of the research done for the wine industry.

Most of the program of the day was related to the same posters presented. So in addition to seeing and commenting particularly with authors throughout the day, there were four general sessions where experts presented them in summary. In these four sessions grouping of posters was based on their areas.

Robert Savé and Anna Puig

Lluís Tolosa taking notes of one poster.

Robert Savé (researcher of IRTA) presented and moderate the session of Viticulture. Olga Busto (professor of URV) did the same with the Oenological Chemistry and Technology session. Anna Puig (researcher of INCAVI) did the same in session of Microbiology in Enology and Viticulture, and finally the sociologist and writer Lluís Tolosa presented and moderate the session of other subjects related to the vine and wine, such as the issues of effects on health, environmental impact, socioeconomic aspects like tourism, food industry or other applications, or historical and cultural aspects and even prehistoric.

The collection of papers presented showed good international quality of research conducted in oenology and viticulture in Catalonia, and at the same time, the wide variety of topics. With the abstracts of papers a book has been edited: “1st Research Conference on Enology and Viticulture in Catalonia, Book of abstracts of the conference organized by CEICS, Tarragona June 4, 2013”, which has been published by Service of the Publications URV, with ISBN 978-84-695-7878-0.

In addition, the conference included the inaugural lesson of the internationally renowned researcher in vine genetics, José Miguel Martínez Zapater, who has an extensive curriculum research in plant genetics.

Since 1998 Dr. Zapater works on the genetics of Vitis, having achieved important results, both on varietal identification by molecular techniques and also on vine transcriptomics, with the study of gene expression in function on the conditions of climate change. Since 2010 is the director of the new Instituto de Ciencias de la Vid y el Vino (ICVV) in Logroño.

The lesson of Dr. Zapater was an excellent review of the genetic variability of Vitis and of the molecular techniques to identify different vine varieties.

We can say that the Conference, with about 70 attendees, was a success. There were researchers, students, technicians and professionals, of URV, INCAVI, IRTA, VITEC, UAB, UB, UPC, UDL, CSIC, and seven companies. In a survey that we asked them to fill up, we have concluded that most of the participants were very satisfied with the conference, its organization, its development, and express their wishdom to do it again, annualy or every two years.